The quasi species nature of a human immunodeficiency virus type 1 (HIV-1) infection coupled with the virus's ability to rapidly mutate away from selective drug pressure has led to the use of multiple drugs in combination as the accepted paradigm for treating systemic HIV-1 infections. Therefore it is quite likely that to obtain a safe and effective anti-HIV-1 microbicide a similar approach will be employed. The long-term objectives this "Integrated preclinical program for topical microbicides", is to provide data that will help justify the clinical development and commercialization of polyethylene-hexamethylene biguanide (PEHMB or PEHMB-derived product) alone or as part of a combination regimen. PEHMB is a potent inhibitor of HIV-1 with minimal toxicity yielding a therapeutic index >1400. Mechanism of action studies has provided evidence that PEHMB's antiviral activity is due to interference with virus binding to the cellular receptors CXCR4 and CCR5. In addition, large-scale production of PEHMB is estimated to be extremely inexpensive (dollars/kg), and we have found PEHMB to be extremely compatible with and stable in normal vaginal gel viscosity building agents. All of these attributes are promising enough to warrant further preclinical evaluation of this compound. In preliminary studies we have also combined PEHMB with members of other classes of putative anti-HIV-1 microbicides including a sulfated dendrimer developed by Dr. Shengrund at Penn State College of Medicine. We propose in this Program to identify the best PEHMB-based combination of drug candidates to maximize the suppressive effect on HIV-1 transmission. This program will involve three research projects integrated into one program. Project I will continue to refine antiviral efficacy and cellular toxicity parameters of the PEHMB scaffold based on computational approaches coupled to iterative rounds of feedback from our biochemical and biological assay systems. In addition, in Project I we will purify PEHMB, work to understand its structure, perform pharmacokinetic and murine toxicity studies as well as begin pre-formulation and formulation work for use in in vivo experimentation and manufacturing schemes. Project II will have the important task of assessing the relative toxicity of PEHMB utilizing both in vitro and in vivo model systems while at the same time provide the intellectual lead in our understanding of the molecular mechanism(s) b y which the PBG class of compounds inhibit virus replication and elicit cellular toxicity. Project II will also examine the impact of microbicides on the intra-vaginal and cervical immune response utilizing well-defined animal models. In Project Ill, we will use state of the art in vitro and in vivo model systems to profile the antiviral efficacy of our lead compound or combination. Project III will examine preformulation parameters, and test the efficacy of the formulated products in vitro including systematic studies to develop combination microbicides based on PEHMB and then test these formulated combinations using xenograph model systems developed by the investigators. All of the efforts outlined in the three projects are focused on providing information needed to move PEHMB and its viable combinations from a pre-clinical to a clinical stage in the drug discovery/development pipeline, with the goal of offering a new class of compounds with specific mode of action distinct from other compounds currently in clinical trials.